Synaptotagmin I (Syt I) is a calcium-binding synaptic vesicle protein that functions as the calcium sensor for neurotransmitter release. Several studies have also implicated Syt I in synaptic vesicle recycling, but its critical role in neurotransmitter release has limited study of Syt I function during endocytosis. The Davis laboratory has recently developed a method to acutely inactivate Syt I following synaptic vesicle release in vivo; this technique, when combined with live imaging of endocytosis, provides a powerful new approach for the study of Syt I function during endocytosis. The experiments presented here will combine these techniques with ultrastructural analyses to define the role of Syt I in endocytosis. First, electron microscopy of the presynaptic terminal will be used to assess the effects of acute inactivation of Syt I during endocytosis. These studies will help determine where in the endocytic cycle Syt I functions. Second, Syt I mutation and deletion constructs will be expressed in fly motor neurons, to identify the portions of the Syt I protein that are required for its endocytic activity. Finally, these tools of analysis will be applied to stoned, a gene with a syt l-like phenotype, to determine if and how their protein products cooperate during endocytosis.